Site-directed mutagenesis of cis-regulatory elements in the 5' flanking region of the rat pyruvate kinase M gene revealed that two out of the three GC boxes (-133/-124 and -48/-39) are involved in the stimulation of a core promoter (-35/+46). These two regions were also protected in DNaseI footprinting assays. Sp1 and Sp3 were identified as binding proteins to all three GC boxes by supershift experiments. Cotransfections in Drosophila SL2 cells revealed a strong stimulatory function of Sp1 and a synergistic effect of Sp3 to Sp1 in the activation of the pyruvate kinase M promoter. No inhibitory effect of Sp3 was detected. These data indicate that binding of Sp1 at two GC boxes is required for full promoter activity of the pyruvate kinase M gene and thus contributes to the observed cell-cycle-dependent expression of this enzyme in proliferating rat thymocytes.